Dendrimer-Based Coatings on a Photonic Crystal Surface for Ultra-Sensitive Small Molecule Detection

We propose and demonstrate dendrimer-based coatings for a sensitive biochip surface that enhance the high-performance sorption of small molecules (i.e., biomolecules with low molecular weights) and the sensitivity of a label-free, real-time photonic crystal surface mode (PC SM) biosensor. Biomolecule sorption is detected by measuring changes in the parameters of optical modes on the surface of a photonic crystal (PC). We describe the step-by-step biochip fabrication process. Using oligonucleotides as small molecules and PC SM visualization in a microfluidic mode, we show that the PAMAM (poly-amidoamine)-modified chip’s sorption efficiency is almost 14 times higher than that of the planar aminosilane layer and 5 times higher than the 3D epoxy-dextran matrix. The results obtained demonstrate a promising direction for further development of the dendrimer-based PC SM sensor method as an advanced label-free microfluidic tool for detecting biomolecule interactions. Current label-free methods for small biomolecule detection, such as surface plasmon resonance (SPR), have a detection limit down to pM. In this work, we achieved for a PC SM biosensor a Limit of Quantitation of up to 70 fM, which is comparable with the best label-using methods without their inherent disadvantages, such as changes in molecular activity caused by labeling.


Photonic Crystal Surface Mode Detection System
Registration of the biomolecule interaction process on the surface was performed in real time using an "EVA 2.0" microfluidic label-free PC SW biosensor (PCbiosensors.com accessed on 22 April 2023, Russia) [21,22,26]. The sensitive surface was a final layer of silicon oxide in a one-dimensional photonic crystal (1D PC). The following 1D PC structure was designed using the impedance approach [57,58] and was used in the experiments: (BK-7-substrate)/H (LH) 2 L /(water), where L is a SiO 2 layer with a thickness of d1 = 215.8 nm, H is a TiO 2 layer with d2 = 70 nm, and L is a SiO 2 layer with d3 = 369.7 nm. The SiO 2 /TiO 2 6-layer structure (started from TiO 2 and finished by the SiO 2 layers) was produced through ion-assisted e-beam deposition.
The optical scheme of the EVA 2.0 biosensor was based on angle interrogation of a PC SM. A stabilized laser beam detected both the PC surface mode (by s-polarization) and the critical angle total internal reflection (by p-polarization). The optical surface mode resonance angular interrogation measured the thickness of the adsorbed layer, and a simultaneous detection of the critical angle total internal reflection provided independent data on the liquid's refractive index.

PC Activation and Functionalization
Before activation and functionalization, PC chips were thoroughly cleaned of organic contaminants according to [26]. The PC chips were submersed in Hellmanex detergent (Hellma, Muellheim, Germany), ultrasonicated with double-distilled water and ethanol three times for 10 min, and dried under nitrogen flow. Next, the cleaned PC chips were treated in a Zepto W6 plasma cleaner (13.56 MHz/100 W, Diener Electronic, Ebhausen, Germany) for 10 min at 600-800 mbar air pressure to create silanol and siloxane groups on the sensitive PC layer.
After cleaning, the PCs were immersed in APTES solution (3%, v/v) for 3 min, rinsed with double-distilled water, and baked for 30 min at 120 • C to remove the moisture (dehydration) present on the PC surface. Quality control of the silanization process was carried out using atomic force microscopy (AFM) (see Section 2.5).

Functionalization of the PC chip
After the cleaning and activation (silanization) procedure, the PC chips were immediately functionalized with PAMAMs ( Figure 1). We used 4.0-generation PAMAMs in all the experiments (PAMAM G4). treated in a Zepto W6 plasma cleaner (13.56 MHz/100 W, Diener Electronic, Ebhausen, Germany) for 10 min at 600-800 mbar air pressure to create silanol and siloxane groups on the sensitive PC layer.
After cleaning, the PCs were immersed in APTES solution (3%, v/v) for 3 min, rinsed with double-distilled water, and baked for 30 min at 120 °C to remove the moisture (dehydration) present on the PC surface. Quality control of the silanization process was carried out using atomic force microscopy (AFM) (see Section 2.5).

Functionalization of the PC chip
After the cleaning and activation (silanization) procedure, the PC chips were immediately functionalized with PAMAMs ( Figure 1). We used 4.0-generation PAMAMs in all the experiments (PAMAM G4). Activated PC chips were treated with a 0.1% solution of GA in water for 10 min, rinsed with water to remove excess GA, and dried under nitrogen flow. After that, PA-MAM G4 1% solution in PBS at pH 7.2 was applied on the GA-treated PC chips for 10 min, rinsed with PBS at pH 7.2, treated again with GA (molar ratio of PAMAM G4:GA was 1:32) for 5 min, and rinsed with water. Prepared PAMAM G4-GA PC chips were kept in PBS at pH 7.2 until used in experiments.
Functionalization with Biotinylated PAMAM G4 (Second Method) Biotinylated PAMAM G4 (PAMAM G4-biotin) was synthesized as reported in [59] with minor modifications. PAMAM G4 in 100 µL of stock solution was dissolved in 100 mM sodium bicarbonate buffer (pH 9.0) to a mass concentration of 0.1%, and NHS-biotin was added to create molar ratios of 1:8 and 1:64. The mixture was stirred for 24 h at RT and then dialyzed against water to remove unconjugated biotin for 12 h at 4 °C.
PC chip modification with PAMAM G4-biotin was carried out in a microfluidic flow cell of the PC SM biosensor. GA (0.1% solution in PBS, pH 7.2), followed by PAMAM G4biotin in PBS at pH 7.2, was run through a flow cell of the bio sensor at a flow rate of 50 µL/min until the signal plateaued (app. 10 min for each stage). The system was thoroughly rinsed with PBS solution after each stage. Activated PC chips were treated with a 0.1% solution of GA in water for 10 min, rinsed with water to remove excess GA, and dried under nitrogen flow. After that, PAMAM G4 1% solution in PBS at pH 7.2 was applied on the GA-treated PC chips for 10 min, rinsed with PBS at pH 7.2, treated again with GA (molar ratio of PAMAM G4:GA was 1:32) for 5 min, and rinsed with water. Prepared PAMAM G4-GA PC chips were kept in PBS at pH 7.2 until used in experiments.

Functionalization with Biotinylated PAMAM G4 (Second Method)
Biotinylated PAMAM G4 (PAMAM G4-biotin) was synthesized as reported in [59] with minor modifications. PAMAM G4 in 100 µL of stock solution was dissolved in 100 mM sodium bicarbonate buffer (pH 9.0) to a mass concentration of 0.1%, and NHS-biotin was added to create molar ratios of 1:8 and 1:64. The mixture was stirred for 24 h at RT and then dialyzed against water to remove unconjugated biotin for 12 h at 4 • C.
PC chip modification with PAMAM G4-biotin was carried out in a microfluidic flow cell of the PC SM biosensor. GA (0.1% solution in PBS, pH 7.2), followed by PAMAM G4-biotin in PBS at pH 7.2, was run through a flow cell of the bio sensor at a flow rate of 50 µL/min until the signal plateaued (app. 10 min for each stage). The system was thoroughly rinsed with PBS solution after each stage.  Detection of 0.05 mg/mL STP in PBS at pH 7.2 with the PAMAM-GA-modified PC chip surface was carried out in the same way as BSA detection.

Low-Molecular-Weight Biomolecule Detection
Some oligonucleotides previously designed for Mycobacterium tuberculosis spacer oligonucleotide typing (spoligotyping) were used to test the ability of the PC SM biosensor to detect small molecules.
Thus, in the Z36-S36 pair, the Z36 oligonucleotide (oligosensor) was the sequence for detecting the model ssDNA target of S36 (oligotarget), and in the Z37-S37 pair, the Z37 oligonucleotide was the sequence for detecting the model ssDNA target S37 sequence. The probe sequences of the oligosensor were biotinylated, which enabled specific binding to the sensitive surface of the PC chip. All oligonucleotides were used at a concentration of 25 pM/mL in PBS at pH 7.2.
STP (0.05 mg/mL in PBS) was incubated with a biotinylated oligosensor for 10 min at RT for complex preparation (STP-biotinylated oligosensor). STP (0.05 mg/mL in PBS), a STP-biotinylated oligosensor, a BSA blocking agent (0.1 mg/mL in PBS), and a biotinylated oligosensor or oligotarget were consecutively run through a microfluidic flow cell with one of the PAMAM-G4-modified PC chips at a flow rate of 50 µL/min until the signal plateaued, at which point the system was thoroughly rinsed with PBS solution.

Atomic Force Microscopy
All images were obtained with an AFM NTEGRA PRIMA I (NT-MDT, Moscow, Russia), and CS37 B cantilevers (NT-MDT, Russia) were used. The cantilevers had a 0.1 N/m force constant, a 16 kHz resonant frequency in an air medium, and an 8 nm tip radius. The semicontact mode scanning method was used, and AFM studies were conducted in a liquid medium (Milli-Q water) to prevent PAMAM damage and deformation upon drying. Substrate modal tilt was removed with Fit Line X filters. AFM measurements were performed directly on a PC.

Results
The method based on the label-free optical detection of biomolecule interactions using a photonic crystal surface mode (PC SW) biosensor allows for real-time tracking of molecule interactions, meaning conclusions about the interaction affinity can be drawn [22]. The previously proposed approach based on creating a 3D dextran matrix on a 1D photonic crystal (PC) surface allowed us to achieve a 20% increase in sorption capacity [26]. Nevertheless, the transition to multiplex detection based on the imaging mode of PC biosensing [20] requires a much larger sorption capacity to detect small molecules, i.e., up to the pM range.
In this study, a poly-amidoamine dendrimer of generation 4.0 (PAMAM G4) was chosen to create a 3D matrix on a PC surface. Using glutaraldehyde (GA) as a crosslinking agent, individual dendrimer molecules were bound to the PC chip surface and conjugated into complex structures with each other, as confirmed by the results of atomic force microscopy (AFM) in Section 3.5. Thus, the measured height of the dendrimer molecule was 4.5 nm [60], and the average layer thickness was 70 nm, with individual conjugates reaching up to 120 nm (see Section 3.5).

Model Protein-Binding Capacity on the PAMAM G4-GA PC Chip
The PC surface was activated, and (3-Aminopropyl)triethoxysilane (APTES) was applied to create a layer with terminal amino groups (see Section 2.3.1). The next step was GA treatment, which could proceed via two routes: (1) involving two dialdehyde carbonyl groups and two adjacent silane amino groups in the formation of Schiff bases or (2) involving one silane amino group and one glutaraldehyde carbonyl group, with the second amine group remaining free and reactive. The second mechanism provided an option to further modify the PC surface using PAMAM G4 with terminal amino groups. The use of PAMAM G4 and GA at a molar ratio of 1:32 PAMAM G4:GA led both to inter-dendrimer interaction with the formation of imine bonds and to the appearance of terminal carbonyl groups capable of further interaction.
In order to evaluate the specific binding of the model protein to the PC chip's PAMAM-G4-functionalized surface, we studied the change in the PC-based biosensor's surface layer thickness at successive stages of its surface pretreatment and in a model experiment. Bovine serum albumin (BSA) was used as a model protein to detect binding to the PAMAM G4-GA layer of the PC chip. Experiments with BSA were carried out in PBS at pH 7.2. BSA solution (0.1 mg/mL) was run through the flow cell at a rate of 50 µL/min until signal stabilization and then rinsed with PBS for 2 min. Figure 2a,b show the sensorgrams (change in the adlayer thickness as a function of time) of BSA binding to the PC surface functionalized with PAMAM G4-GA (outer PC surface). For comparison, we used a method of modifying the PC chip surface with APTES [26]. The data obtained show a 14-fold increase in the sorption capacity of the PC sensitive layer modified with PAMAM G4-GA compared to the APTES-modified PC ( Table 2).

Model Protein-Binding Capacity on the PAMAM G4-GA PC Chip
The PC surface was activated, and (3-Aminopropyl)triethoxysilane (APTES) was applied to create a layer with terminal amino groups (see Section 2.3.1). The next step was GA treatment, which could proceed via two routes: (1) involving two dialdehyde carbonyl groups and two adjacent silane amino groups in the formation of Schiff bases or (2) involving one silane amino group and one glutaraldehyde carbonyl group, with the second amine group remaining free and reactive. The second mechanism provided an option to further modify the PC surface using PAMAM G4 with terminal amino groups.
The use of PAMAM G4 and GA at a molar ratio of 1:32 PAMAM G4:GA led both to inter-dendrimer interaction with the formation of imine bonds and to the appearance of terminal carbonyl groups capable of further interaction.
In order to evaluate the specific binding of the model protein to the PC chip's PA-MAM-G4-functionalized surface, we studied the change in the PC-based biosensor's surface layer thickness at successive stages of its surface pretreatment and in a model experiment. Bovine serum albumin (BSA) was used as a model protein to detect binding to the PAMAM G4-GA layer of the PC chip. Experiments with BSA were carried out in PBS at pH 7.2. BSA solution (0.1 mg/mL) was run through the flow cell at a rate of 50 µL/min until signal stabilization and then rinsed with PBS for 2 min. Figure 2a,b show the sensorgrams (change in the adlayer thickness as a function of time) of BSA binding to the PC surface functionalized with PAMAM G4-GA (outer PC surface). For comparison, we used a method of modifying the PC chip surface with APTES [26]. The data obtained show a 14-fold increase in the sorption capacity of the PC sensitive layer modified with PAMAM G4-GA compared to the APTES-modified PC ( Table 2).

Streptavidin Binding Capacity on the PAMAM G4-GA PC Chip
Next, we evaluated the specific binding of STP to the PAMAM-G4-functionalized surface of the PC chip. STP was the key component of the small molecule detection complex. Experiments with STP (0.05 mg/mL) were carried out in the same way as with BSA (Section 3.1).
The fabrication of a 3D PAMAM-G4-GA-based structure on the PC chip surface made it possible to significantly increase the sorption capacity of biomolecules. In the case of protein detection, such as with STP or avidin, the 64 terminal amino groups in the structure of PAMAM G4 made it possible both to increase the sorption capacity of the PC

Streptavidin Binding Capacity on the PAMAM G4-GA PC Chip
Next, we evaluated the specific binding of STP to the PAMAM-G4-functionalized surface of the PC chip. STP was the key component of the small molecule detection complex. Experiments with STP (0.05 mg/mL) were carried out in the same way as with BSA (Section 3.1).
The fabrication of a 3D PAMAM-G4-GA-based structure on the PC chip surface made it possible to significantly increase the sorption capacity of biomolecules. In the case of protein detection, such as with STP or avidin, the 64 terminal amino groups in the structure of PAMAM G4 made it possible both to increase the sorption capacity of the PC surface and to increase the sorption specificity by covalently binding a biotin molecule to each PAMAM G4 terminal amino group.
The sorption capacity of PAMAM G4-biotin prepared at different molar ratios was also investigated. The sensorgrams of STP bound to the PAMAM-G4-biotin-modified PC surface at molar ratios 1:8 and 1:64 of PAMAM G4:biotin are shown in Figures 3 and 4, respectively. surface and to increase the sorption specificity by covalently binding a biotin molecule to each PAMAM G4 terminal amino group.
The sorption capacity of PAMAM G4-biotin prepared at different molar ratios was also investigated. The sensorgrams of STP bound to the PAMAM-G4-biotin-modified PC surface at molar ratios 1:8 and 1:64 of PAMAM G4:biotin are shown in Figures 3 and 4, respectively.   Here, we see that an eight-fold increase in the biotinylating degree of PAMAM G4 resulted in an almost eight-fold increase in the amount of bound STP, i.e., the increase was completely specific. Both the minimum and maximum biotinylating degrees of PAMAM G4 provided a greater amount of bound protein than the planar PC surface modified with biotin ( Figure 5, Table 3). Here, we see that an eight-fold increase in the biotinylating degree of PAMAM G4 resulted in an almost eight-fold increase in the amount of bound STP, i.e., the increase was completely specific. Both the minimum and maximum biotinylating degrees of PAMAM G4 provided a greater amount of bound protein than the planar PC surface modified with biotin ( Figure 5, Table 3).  Thus, we determined that the method for PC surface modification that provided the maximum sorption of proteins (on the examples of BSA and STP) was PAMAM G4-biotin with a molar ratio of 1:64.

Detection of Low-Molecular-Weight Biomolecule Interaction with PC SM Biosensor
The main task was to evaluate the possibility of detecting small biomolecule sorption and interaction using a PC SM biosensor. For this, STP-biotinylated oligosensor complexes (STP-biotinylated Z36, STP-biotinylated Z37) were prepared and bound to the surface of a PAMAM-G4-biotin-modified PC chip.
The detection capacity of oligonucleotide sequence interaction was carried out with the PAMAM-G4-biotin (molar ratio of 1:64)-modified PC chip. A complex STP-biotinylated oligosensor was prepared and bound to the PAMAM G4-biotin PC chip. For comparison, we used PCs modified with APTES and with PAMAM G4-GA. Figures 6-8 show the sorption curves (sensorgrams) of the oligosensor-STP complex-the oligosensor and its complementary oligotarget on the PC surface-modified with PAMAM G4-GA, PAMAM G4-biotin, and APTES, respectively. Comparative data are given in Table 4. The greatest oligotarget increase was obtained for the PAMAM-G4-GA-modified surface and could be explained by nonspecific binding to the PAMAM-G4-GA-modified PC surface.  Thus, we determined that the method for PC surface modification that provided the maximum sorption of proteins (on the examples of BSA and STP) was PAMAM G4-biotin with a molar ratio of 1:64.

Detection of Low-Molecular-Weight Biomolecule Interaction with PC SM Biosensor
The main task was to evaluate the possibility of detecting small biomolecule sorption and interaction using a PC SM biosensor. For this, STP-biotinylated oligosensor complexes (STP-biotinylated Z36, STP-biotinylated Z37) were prepared and bound to the surface of a PAMAM-G4-biotin-modified PC chip.
The detection capacity of oligonucleotide sequence interaction was carried out with the PAMAM-G4-biotin (molar ratio of 1:64)-modified PC chip. A complex STP-biotinylated oligosensor was prepared and bound to the PAMAM G4-biotin PC chip. For comparison, we used PCs modified with APTES and with PAMAM G4-GA. Figures 6-8 show the sorption curves (sensorgrams) of the oligosensor-STP complex-the oligosensor and its complementary oligotarget on the PC surface-modified with PAMAM G4-GA, PAMAM G4-biotin, and APTES, respectively. Comparative data are given in Table 4. The greatest oligotarget increase was obtained for the PAMAM-G4-GA-modified

Sensor Baseline Noise, LoD, LoQ, and Dynamic Range
The baseline noise, or standard deviation of the baseline thickness (STD (da)), current biosensor was δd = 2 × 10 −4 nm for thin, tight adlayers (such as the biotin-mo PC surface in Figure 5), while the baseline noise for thick, loose adlayers (such as ures 4 and 6) was 6 × 10 −4 nm. To estimate the sensitivity of the biosensor, the latt was used. We defined the Limit of Detection (LoD) as 3 × the standard deviation baseline and the Limit of Quantitation (LoQ) as 10 × the standard deviation of the ba In Figure 4, the injection of a streptavidin concentration of 0.05 mg/mL led to a thi increase of 15 nm (see Table 3), resulting in the S/N ratio of 25,000 (15 nm/0.000 Therefore, in this case, the LoQ for streptavidin in the PAMAM G4-biotin layer pg/mL (0.05 [mg/mL]/2500). On the other hand, in Figure 6, the concentration of ol cleotides at 25 pM/mL resulted in a thickness increase of 2.1 nm (see Table 4), giv S/N ratio of 3500 (2.1 nm/0.0006 nm). The LoQ for oligonucleotides in the PAMA GA layer was 70 fM/mL (25 [pM/mL]/350).
The dynamic range (DR) of the current biosensor was limited by the size of th sor's matrix. The 25 nm shift in Figure 4 amounted to ~14% of the DR, indicating t overall operating range of the biosensor in terms of adsorption thickness was ap mately 180 nm.

Atomic Force Microscopy
Atomic force microscopy (AFM) was chosen as an independent method for s investigation at various stages of PC chip formation and biomolecule detection. To c the quality of the APTES-treated PC sensitive surface (see Section 2.3.1), we cre scratch and measured the thickness of the APTES coating as we described earlie AFM studies were conducted in a liquid medium (Milli-Q water) to prevent the PA G4 layer from damage and deformation upon drying. Figure 9 illustrates the topo

Sensor Baseline Noise, LoD, LoQ, and Dynamic Range
The baseline noise, or standard deviation of the baseline thickness (STD (d a )), for the current biosensor was δd = 2 × 10 −4 nm for thin, tight adlayers (such as the biotinmodified PC surface in Figure 5), while the baseline noise for thick, loose adlayers (such as in Figures 4 and 6) was 6 × 10 −4 nm. To estimate the sensitivity of the biosensor, the latter STD was used. We defined the Limit of Detection (LoD) as 3 × the standard deviation of the baseline and the Limit of Quantitation (LoQ) as 10 × the standard deviation of the baseline. In Figure 4, the injection of a streptavidin concentration of 0.05 mg/mL led to a thickness increase of 15 nm (see Table 3), resulting in the S/N ratio of 25,000 (15 nm/0.0006 nm). Therefore, in this case, the LoQ for streptavidin in the PAMAM G4biotin layer was 20 pg/mL (0.05 [mg/mL]/2500). On the other hand, in Figure 6, the concentration of oligonucleotides at 25 pM/mL resulted in a thickness increase of 2.1 nm (see Table 4), giving the S/N ratio of 3500 (2.1 nm/0.0006 nm). The LoQ for oligonucleotides in the PAMAM G4-GA layer was 70 fM/mL (25 [pM/mL]/350).
The dynamic range (DR) of the current biosensor was limited by the size of the sensor's matrix. The 25 nm shift in Figure 4 amounted to~14% of the DR, indicating that the overall operating range of the biosensor in terms of adsorption thickness was approximately 180 nm.

Atomic Force Microscopy
Atomic force microscopy (AFM) was chosen as an independent method for surface investigation at various stages of PC chip formation and biomolecule detection. To control the quality of the APTES-treated PC sensitive surface (see Section 2.3.1), we created a scratch and measured the thickness of the APTES coating as we described earlier [26]. AFM studies were conducted in a liquid medium (Milli-Q water) to prevent the PAMAM G4 layer from damage and deformation upon drying. Figure 9 illustrates the topology of the scratches made on different PCs modified with APTES, APTES-PAMAM G4-biotin, and APTES-GA-PAMAM G4. The scanning area was 10 × 10 um, and the number of points was 256 × 256. The scan rate was 1 Hz. Height histograms allowed us to measure coating thickness [32]. As seen in the histogram in Figure 9a, the mean thickness of the APTES layer was 3.5 nm, and the mean height of roughness was 0.5 nm. In agreement with the data given in [61], we assume that the silanization protocol chosen to modify the PC chip made it possible to obtain uniform coatings with a reproducible surface topology. the scratches made on different PCs modified with APTES, APTES-PAMAM G4-biotin, and APTES-GA-PAMAM G4. The scanning area was 10 × 10 um, and the number of points was 256 × 256. The scan rate was 1 Hz. Height histograms allowed us to measure coating thickness [32]. As seen in the histogram in Figure 9a, the mean thickness of the APTES layer was 3.5 nm, and the mean height of roughness was 0.5 nm. In agreement with the data given in [61], we assume that the silanization protocol chosen to modify the PC chip made it possible to obtain uniform coatings with a reproducible surface topology. The mean height of the PAMAM G4-biotin coating was 18 nm, and the mean height of roughness was 5.3 nm (Figure 9b). Results for the PAMAM-GA layer on the modified PC surface are shown in Figure 9c. The mean thickness was 70 nm, and the mean height of roughness was 9.5 nm. The results obtained are consistent with the data from [56,57]. Authors have shown that generations of PAMAMs with lower molecular weights have smaller heights [62,63]. Therefore, generation 4.0 of PAMAM should have molecules of about 10 nm in height, which means that our data agree with these works.

Discussion
The PC surface modification approach proposed in this article significantly increased PC chip sorption capacity. In general, we could say that, in comparison to a silane-modified planar PC surface, the response was 8-10 times greater both for relatively large biomolecules, such as proteins, and relatively small biomolecules, such as oligonucleotides. This work is a continuation of a previous paper published in this journal [26]. The thickness of PAMAM-based coatings measured using AFM (18 and 70 nm) was comparable to that of 3D ED-matrix-based coatings (range of 10-40 nm), but the values of sorption capacity compared to previously developed 3D polysaccharide coatings on PC surfaces increased by 5-6 times.
The choice of dendrimer generation was based on known [44] considerations that structural flexibility is important for the formation of dendrimer complexes with DNA molecules, the reduction of which occurs at higher (five and more) generations of PA-MAMs. The assumption of increased sorption capacity at higher dendrimer generations due to a much greater number of terminal amino groups was rejected on the basis that steric considerations do not allow all capable groups to participate in binding. This, however, requires further investigation.
Here, we proposed two approaches to PC surface modification. The first approach consisted of the creation of a 3D matrix of PAMAM G4 globules crosslinked with GA on a PC sensitive surface. However, despite all efforts, this matrix formation approach had a stochastic character, as confirmed with AFM by the presence of aperiodic large clusters on the PC. The mean height of the PAMAM G4-biotin coating was 18 nm, and the mean height of roughness was 5.3 nm (Figure 9b). Results for the PAMAM-GA layer on the modified PC surface are shown in Figure 9c. The mean thickness was 70 nm, and the mean height of roughness was 9.5 nm. The results obtained are consistent with the data from [56,57]. Authors have shown that generations of PAMAMs with lower molecular weights have smaller heights [62,63]. Therefore, generation 4.0 of PAMAM should have molecules of about 10 nm in height, which means that our data agree with these works.

Discussion
The PC surface modification approach proposed in this article significantly increased PC chip sorption capacity. In general, we could say that, in comparison to a silanemodified planar PC surface, the response was 8-10 times greater both for relatively large biomolecules, such as proteins, and relatively small biomolecules, such as oligonucleotides. This work is a continuation of a previous paper published in this journal [26]. The thickness of PAMAM-based coatings measured using AFM (18 and 70 nm) was comparable to that of 3D ED-matrix-based coatings (range of 10-40 nm), but the values of sorption capacity compared to previously developed 3D polysaccharide coatings on PC surfaces increased by 5-6 times.
The choice of dendrimer generation was based on known [44] considerations that structural flexibility is important for the formation of dendrimer complexes with DNA molecules, the reduction of which occurs at higher (five and more) generations of PAMAMs.
The assumption of increased sorption capacity at higher dendrimer generations due to a much greater number of terminal amino groups was rejected on the basis that steric considerations do not allow all capable groups to participate in binding. This, however, requires further investigation.
Here, we proposed two approaches to PC surface modification. The first approach consisted of the creation of a 3D matrix of PAMAM G4 globules crosslinked with GA on a PC sensitive surface. However, despite all efforts, this matrix formation approach had a stochastic character, as confirmed with AFM by the presence of aperiodic large clusters on the PC.
The second approach, based on the preliminary specific biotinylation of PAMAM G4 terminal amino groups, proved to be a reliable and reproducible method for fabricating effective sensor biochips.
The achieved response values (∆d = 1.5-2 nm) during oligonucleotide detection on the sensorgrams gave us the possibility to detect concentrations of an order of magnitude lower than those used. In combination with the achieved detection time (3-5 min), this allowed us to classify the method as ultra-sensitive and fast.

Conclusions
In this work, we proposed approaches for PC sensitive surface functionalization with dendrimers. We developed and studied PAMAM-G4-based PC coatings. The resulting 3D structures on the PC surface provided an increase in sorption capacity of over 10 times compared to that of the PC planar surface, as well as more than 5-6 times compared to 3D structures based on dextran with different anchor groups. The proposed methods of PC surface modification showed their effectiveness both for large molecules, such as proteins, and for small molecules, such as oligonucleotides.